Jill E. Ogden

Inhibition of nitric oxide synthase — potential for a novel class of therapeutic agent?

The free-radical gas nitric oxide (NO) plays an important role in a wide and diverse range of physiological processes. As progress is made in understanding the biological function of NO, there is growing interest in the possibility that inhibitors of NO synthase (NOS) may be of clinical use in the therapy of certain disease states. The search for novel and clinically relevant inhibitors of this enzyme represents a truly multidisciplinary approach to drug screening and will no doubt benefit from the application of recombinant DNA (rDNA) technology.

Haemoglobin-based red cell substitutes: current status.

Chemically modified haemoglobin solutions represent a potential alternative to the transfusion of donor blood. The theoretical advantages of these products include an oxygen delivery potential greater than that of conventional plasma expanders, prolonged shelf‐life, universal compatibility and the absence of pathogenic viruses. Principal concerns have been safety issues including renal toxicity, coagulopathy and vasoactivity. The proposed indications for these solutions are primarily resuscitation of patients in haemorrhagic shock and perioperative haemodilution during elective surgery. Three products have now undergone phase I safety trials in human subjects and phase II safety and efficacy trials are planned in the near future.

Cloning and characterisation of the Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter

The Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase (GUT2) promoter and part of the protein-coding region have been isolated on a 6.3-kb genomic DNA fragment. Nucleotide sequence analysis shows that the promoter has many structural features in common with yeast glycolytic enzyme promoters. Chromosomal mapping indicates that this genomic fragment is located on chromosome XII. The GUT2 promoter has been used to construct a recombinant human albumin (reHA) secretion vector; yeast transformed with this vector secrete reHA into the culture supernatant.

High level synthesis and secretion of human urokinase using a late gene promoter of the Autographa californica nuclear polyhedrosis virus

A cDNA encoding human urokinase was inserted into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome at the polyhedrin gene locus under control of a duplicated copy of the late, basic protein gene promoter. The insect-derived urokinase was produced predominantly in the form of single-chain, pro-urokinase, with a molecular mass of 50 kDa, and demonstrated fibrinolytic activity. Synthesis and secretion of urokinase was first detected at 6 hours post-infection and continued steadily throughout the infection period. Comparisons with urokinase synthesised using the very late AcNPV polyhedrin gene promoter revealed that, although the polyhedrin promoter is intrinsically stronger, the yield of secreted urokinase was higher using the basic protein gene promoter. These data support the hypothesis that the host cell secretory pathway is compromised in the very late stages of baculovirus infection and may provide an explanation for why, in general, secreted and membrane-targeted proteins are not produced to the high levels observed with other proteins, when using very late baculovirus gene promoters.

Production of recombinant human hemoglobin A in Saccharomyces cerevisiae

Publisher Summary The ability to produce human hemoglobin (Hb) in a genetically engineered host microorganism offers a number of valuable opportunities. First, a recombinant microorganism provides an attractive alternative to outdated stocks of red blood cells as a source of Hb for formulation into an Hb-based red cell substitute. Second, specific mutant Hbs can be synthesized and produced in the host for subsequent detailed structural and functional studies. Both Saccharomyces cerevisiae and Escherichia coli have been employed for the production of recombinant human Hb. This chapter presents a straightforward method for producing recombinant Hb (rHb) in S. cerevisiae and E. coli that avoids the refolding and reconstitution steps. It also describes methods for expression of recombinant human Hb A (rHb A) in S. cerevisiae, the purification of the protein, and techniques used for subsequent structural and functional characterization. Coexpression of α- and β-globins to produce rHb A can be achieved either by using a single plasmid carrying both α- and β-globin expression cassettes, or by cotransformation with two plasmids, each carrying α- and β-globin expression cassettes and complementary auxotrophic markers. Examples of both of these approaches are also discussed in the chapter.

Expression of rat neuronal nitric oxide synthase in Saccharomyces cerevisiae.

The rat neuronal nitric oxide synthase (nNOS) cDNA was expressed in Saccharomyces cerevisiae under the control of a hybrid PGK/GAL promoter, PAL. Galactose induction resulted in the production of a soluble 160 kDa protein that was recognised by anti-neuronal NOS antibody. NOS activity was detected by the conversion of [3H]arginine to [3H]citrulline and required the addition of the cofactors, calmodulin and tetrahydrobiopterin. The activity was inhibited by the arginine analogues NG-nitro-L-arginine and NG-nitro-L-arginine methyl ester.

Secretion of single-chain urokinase-type plasminogen activator from insect cells.

A cDNA encoding human urokinase-type plasminogen activator was inserted downstream from the polyhedrin promoter of the baculovirus Autographa californica nuclear polyhedrosis virus. A protein of similar Mr to urokinase (UK) was synthesized and approx. 90% was secreted from recombinant virus-infected Spodoptera frugiperda cells. Zymography and Western blotting analysis of the insect-derived protein demonstrated that it was comprised solely of the high-Mr form of UK. No low-Mr UK was detected. Amidolytic activity assays showed that 96% of the insect cell-derived UK was in the single-chain proenzyme form. The yield of UK from insect cells was 1986 international units/ml/10(6) infected cells.